Project Summary/Abstract of the funded grant (from the original proposal): Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolism, and many other biological processes. The fascination of this phenomenon is to explain how a biochemical mechanism (i) can robustly sustain a long period (~24 h) oscillation whose frequency keeps time so precisely, and (ii) enhance fitness in the natural environment. These questions remain critically important unanswered issues in the circadian rhythms field. For example, the adaptive value is not clear for the most obvious circadian characteristic?a robust self-sustained oscillation in constant conditions. If ?anticipation? of future temporal events (e.g., dawn, dusk, etc.) is the goal of circadian timekeepers, why is a temperature- compensated ?hourglass timer? that is initiated by dawn or dusk not sufficient? And yet evolution ubiquitously selected an oscillator that sustains itself in non-natural continuous as the timekeeper for regulating daily processes, and this characteristic forms a core defining property of circadian rhythms. The overall goal of this project is to determine which characteristics of rhythmic environments provide selective pressures that direct cellular organization of gene expression and metabolism to promote properties of circadian timekeeping. Identifying the selective pressures & evolutionary steps that can lead to biological timekeeping will enable a more profound understanding of circadian mechanisms and the way(s) they might be reinforced to aid human health and performance. The unique characteristics of model systems will be harnessed to attain the goal of this project by a multifaceted approach. First, in free-living organisms, fitness tests, transcriptomics, and biochemistry will determine if metabolic conditions dictate whether sustained oscillators are necessarily adaptive. Second, the temporal dimensions of host/microbiome relations will be manipulated to ascertain if the gut microbiome is under active selection for timekeeping ability. Finally, a novel experimental selection approach will identify which environmental pressures are capable of evolving circadian clocks. The answers to these questions will help us to better understand general principles of fundamental circadian organization and rhythmic regulation of metabolism; this understanding can help us to better design therapies for disorders in which circadian clocks are implicated.